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. 2022 Dec 21;10(6):e0163422.
doi: 10.1128/spectrum.01634-22. Epub 2022 Nov 29.

Long-Term and Low-Level Envelope C2V3 Stimulation by Highly Diverse Virus Isolates Leads to Frequent Development of Broad and Elite Antibody Neutralization in HIV-1-Infected Individuals

Francisco Martin  1 , José Maria Marcelino  1   2 , Claudia Palladino  1 , Inês Bártolo  1 , Susana Tracana  1 , Inês Moranguinho  1 , Paloma Gonçalves  1 , Rita Mateus  1 , Rita Calado  1 , Pedro Borrego  1 , Thomas Leitner  3 , Sofia Clemente  4 , Nuno Taveira  1   2
Affiliations

Long-Term and Low-Level Envelope C2V3 Stimulation by Highly Diverse Virus Isolates Leads to Frequent Development of Broad and Elite Antibody Neutralization in HIV-1-Infected Individuals

Francisco Martin et al. Microbiol Spectr. .

Abstract

A minority of HIV-1-infected patients produce broadly neutralizing antibodies (bNAbs). Identification of viral and host correlates of bNAb production may help develop vaccines. We aimed to characterize the neutralizing response and viral and host-associated factors in Angola, which has one of the oldest, most dynamic, and most diverse HIV-1 epidemics in the world. Three hundred twenty-two HIV-1-infected adults from Angola were included in this retrospective study. Phylogenetic analysis of C2V3C3 env gene sequences was used for virus subtyping. Env-binding antibody reactivity was tested against polypeptides comprising the C2, V3, and C3 regions. Neutralizing-antibody responses were determined against a reference panel of tier 2 Env pseudoviruses in TZM-bl cells; neutralizing epitope specificities were predicted using ClustVis. All subtypes were found, along with untypeable strains and recombinant forms. Notably, 56% of the patients developed cross neutralizing, broadly neutralizing, or elite neutralizing responses. Broad and elite neutralization was associated with longer infection time, subtype C, lower CD4+ T cell counts, higher age, and higher titer of C2V3C3-specific antibodies relative to failure to develop bNAbs. Neutralizing antibodies targeted the V3-glycan supersite in most patients. V3 and C3 regions were significantly less variable in elite neutralizers than in weak neutralizers and nonneutralizers, suggesting an active role of V3C3-directed bNAbs in controlling HIV-1 replication and diversification. In conclusion, prolonged and low-level envelope V3C3 stimulation by highly diverse and ancestral HIV-1 isolates promotes the frequent elicitation of bNAbs. These results provide important clues for the development of an effective HIV-1 vaccine. IMPORTANCE Studies on neutralization by antibodies and their determinants in HIV-1-infected individuals have mostly been conducted in relatively recent epidemics caused by subtype B and C viruses. Results have suggested that elicitation of broadly neutralizing antibodies (bNAbs) is uncommon. The mechanisms underlying the elicitation of bNAbs are still largely unknown. We performed the first characterization of the plasma neutralizing response in a cohort of HIV-1-infected patients from Angola. Angola is characterized by an old and dynamic epidemic caused by highly diverse HIV-1 variants. Remarkably, more than half of the patients produced bNAbs, mostly targeting the V3-glycan supersite in HIV-1. This was associated with higher age, longer infection time, lower CD4+ T cell counts, subtype C infection, or higher titer of C2V3C3-specific antibodies relative to patients that did not develop bNAbs. These results may help develop the next generation of vaccine candidates for HIV-1.

Keywords: Angola; Env diversity; Env-specific antibodies; HIV-1 infection; bNAbs; broadly neutralizing antibodies; neutralizing epitopes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Neutralization potency and breadth per sampling year. (A) Potency of neutralization (percent neutralization at a 1:40 plasma dilution) of samples collected in 2009 and 2014 as assessed against the 12 Env-pseudotyped viruses in the indicator panel. Means and 95% confidence intervals are shown. (B) Neutralization breadth (number of Env-pseudotyped viruses neutralized at >20%) in samples collected in 2009 and 2014. Medians and interquartile ranges are shown. P values were obtained using the Mann-Whitney U test.
FIG 2
FIG 2
NSs of samples from HIV-1-infected patients from Angola as a function of year of sampling and sex. (A) NSs in 2009 is represented in blue and in 2014 in red; NSs for all patients is in green. (B) Angolan patients from 2009 and 2014 were categorized into 4 groups according to the NS as follows: nonneutralizers or weak neutralizers, <6 (gray); cross neutralizers, 6 to 17 (yellow); broad neutralizers, 18 to 24 (orange); elite neutralizers, 25 to 36 (red). (C) NS in matched samples collected in 2009 and 2014, showing the NS categories. (D) NS in males and females. Medians and interquartile ranges are shown. P values were obtained using the Mann-Whitney U test.
FIG 3
FIG 3
Antibody neutralization titers in a subset of plasma samples from elite and broad neutralizers from 2009 (n = 28) and 2014 (n = 10). (A) Heat map of the neutralization titers (ID50) and neutralization scores against the 12 Env-pseudotyped-virus indicator panel. ID50s are color coded, with darker colors indicating higher ID50s. Subtype was determined in the env gene except for samples noted with asterisks, where subtype was determined in the pol gene. +, number of CD4+ T cells determined in 2009; ND, not done due to lack of sample; NA, not available. VSV-pseudotyped viruses were used as a neutralization specificity control. ID50 heat map color code: white, <40; bright yellow, 40; yellow, 80 to 320; orange, 640 to 1,280; red, 2,560. (B) Comparison of antibody neutralization titers in 2009 and 2014. Log10 ID50s obtained with each patient sample against the 12 Env-pseudotyped viruses in the indicator panel are plotted. Lines indicate medians with interquartile ranges. The P value was obtained using the Mann-Whitney U test.
FIG 4
FIG 4
Impact of HIV-1 subtype on antibody neutralization. (A) Neutralization score in patients infected with the two most common subtypes in Angola (in 2009), C (n = 27) and A1 (n = 26), and in patients infected with other subtypes and recombinant forms (n = 56). The Kruskal-Wallis nonparametric test was used to analyze the difference in median NS for all subtypes (P = 0.014). Dunn’s multiple-comparison test was used to analyze differences in NS between subtypes. Medians and interquartile ranges are shown. (B) Percent neutralization of each of the 12 Env-pseudotyped viruses in the indicator panel by the plasma samples (at a 1:40 dilution) from the Angolan patients (n = 236). Mean percent neutralization and 95% confidence interval bars against a given virus from the indicator panel are shown. Statistically significant differences are represented by the P values obtained with Dunn’s multiple-comparison test. ***, P < 0.0001; **, P < 0.001; *, P < 0.05.
FIG 5
FIG 5
Correlation between neutralization score, CD4+ T cell counts, and patient’s age. (A) Neutralization score differences between 2009 patients with CD4+ T cell counts of ≤200/μL at study entry and patients with counts of >200/μL. Median and interquartile range are shown. P values were obtained using the Mann-Whitney U test. (B) Correlation of neutralization score with CD4+ T cell counts in 2009 and 2014. (C) Correlation of neutralization score with patient age in 2009 and 2014. Data for samples collected in 2009 and in 2014 are in blue and in red, respectively. The linear trend is shown with mean and 95% CI bands; Spearman r and P values are indicated.
FIG 6
FIG 6
Cluster analysis and heat map of the predicted epitope specificity in the top neutralizing patients from Angola. At the top of the columns, known bNAb epitopes are colored according to the respective epitope specificities, as shown in the key. The identification of the plasma samples and bNAbs is shown at the bottom. Cluster analysis for both rows and columns was done according to the Pearson correlation (46). Blue colors in the heat map represent lower neutralization activity and red colors higher neutralization activity. Each column represents the neutralization values of a given plasma sample or a bNAb of known specificities against the panel of 12 Env-pseudotyped viruses whose names are on the right. Black triangles indicate samples from 2014.
FIG 7
FIG 7
Association between titer of antibody binding to C2V3C3 recombinant polypeptides of different subtypes and neutralization score (in 2009). Filled symbols represent titers of antibody binding of the broad/elite neutralizers to a given C2V3C3 subtype. Unfilled symbols are the titers of antibody binding of the nonneutralizers/weak neutralizers and cross neutralizers to a given C2V3C3 subtype. Associations were assessed by Spearman analyses. P values and Spearman r values are indicated. Linear trend is shown with mean and 95% CI bands.
FIG 8
FIG 8
Amino acid entropy differences in the C2V3C3 region between neutralization categories. (A) Shannon’s entropy difference between nonneutralizers/weak neutralizers and cross neutralizers. (B) Shannon’s entropy difference between nonneutralizers/weak neutralizers and broad neutralizers. (C) Shannon’s entropy difference between nonneutralizers/weak neutralizers and elite neutralizers. Sites with significant entropy difference (P ≤ 0.05) are shown in red. Gray boxes delimit the V3 region. Numbers on the x axes indicate the amino acid position in HIV-1.
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